This invention relates to methods and reagents for oligonucleotide synthesis, including for instance, reagents used to prepare labeled oligonucleotides.
A variety of approaches exist, and in turn, a variety of reagents are available, for use in incorporating labeled molecules in synthetic oligonucleotides. For instance, the following sections repeat or paraphrase certain relevant portions of Nelson U.S. Pat. No. 5,451,463 in this regard, which itself is referred to in greater detail below.
xe2x80x9cMethods to covalently attach labels and reporter molecules to oligonucleotides have provided valuable tools in the field of molecular biology and gene probe diagnostics. Recent advances in the preparation of non-isotopic gene probes, DNA sequencing (Connell, C. et al. [1987] Biotechniques 5:342-346; Kaiser, R., S. Mackellar, R. Vinayak, J. Sanders, R. Saavedra, L. Hood [1989] Nucleic Acids Res. 17:6087-6102), electron microscopy (Sproat, B. S., B. Beijer, P. Rider [1987] Nucleic Acids Res. 15:6181-6196), and X-ray crystallography (Sproat et al. [1987] Nucleic Acids Res. 15:4837-4848) have provided the impetus for the development and improvement of such methods. Similarly, new and emerging applications employing the polymerase chain reaction (PCR) technology (Hultman, T., S. Bergh, T. Moks, M. Uhlen [1991] Biotechniques 10:84-93; Landgraf, A., B. Reckmann, A. Pingoud [1991] Analytical Biochemistry 193:231-235; Zimran, A., C. Glass, V. Thorpe, E. Beutler [1989] Nucleic Acids Res. 17:7538) have further expanded the need for convenient and versatile reagents to chemically modify oligonucleotides.xe2x80x9d
xe2x80x9cCurrent methods to introduce chemical modifications into oligonucleotides typically employ the use of non-nucleosidic phosphoramidite reagents during automated oligonucleotide synthesis. Such methods, however, are generally limited to single modifications at only the 5xe2x80x2 terminus, since the 3xe2x80x2 terminus remains attached to the solid support. An inherent disadvantage of such methods is that the labeling reagents tend to terminate chain elongation at the point they are introduced (5xe2x80x2 terminus) and therefore only single modifications can be performed. Chemical modifications that have been introduced in this fashion are primary aliphatic amine (Sinha, N. D., R. M. Cook [1988] Nucleic Acids Res. 16:2659-2669) and thiol (Connolly, B. [1985] Nucleic Acids Res. 13:4485-4502) functionalities. Oligonucleotides functionalized with primary aliphatic amines or thiol groups must be subsequently derivatized with labels such as biotin, fluorescein, and enzymes. Such derivatization requires a second reaction and purification step which minimizes the convenience and practicality of this method. Cocuzza expanded this method to directly incorporate a single biotin label into an oligonucleotide at the 5xe2x80x2 terminus (Cocuzza, A. [1989] Tetrahedron Lett. 30:6287-6290).xe2x80x9d
Nelson et al. introduced a new type of non-nucleosidic phosphoramidite reagent that employs a 1,2-ethanediol backbone (Nelson, P., R. Sherman-Gold, R. Leon [1989] Nucleic Acids Res. 17:7179-7186). This reagent allowed primary aliphatic amines to be incorporated multiple times and at any position of the oligonucleotide. The development of this method was said to eliminate the termination of chain elongation during synthesis, an inherent problem of the above method. Employment of the 1,2-ethanediol backbone allowed the phosphoramidite reagent to be incorporated in the same manner as a normal nucleoside phosphoramidite, at any position and multiple times. Misiura et al. expanded the use of the 1,2-ethanediol backbone derived from a glycerol intermediate, to directly incorporate multiple biotin groups into oligonucleotides (Misiura, K., I. Durrant, M. Evans, M. Gait [1990] Nucleic Acids Res. 18:4345-4354). The development of a 1,2-ethanediol backbone modification method was also said to provide better utility and versatility, especially in the field of gene probe diagnostics where multiple labels yield greater signal detection.
A more recent approach, described in U.S. Pat. No. 5,451,463 (Nelson et al.), is said to overcome the above-described disadvantages by providing improved non-nucleosidic reagents to directly modify or label oligonucleotides via automated solid phase synthesis. The ""463 patent provides a trifunctional reagent possessing a primary hydroxyl, a secondary hydroxyl, and a primary amino group. This reagent is said to be useful in solid phase oligonucleotide synthesis for the convenient labeling of the 3xe2x80x2-terminus. The secondary hydroxyl may be a phosphoramidite derivative permitting the attachment to the solid phase support. The reporter molecule may be attached to the trifunctional molecule prior to the completion of the oligonucleotide synthesis or after the oligonucleotide is cleaved from the support.
Finally, U.S. Pat. No. 5,723,591 (Livak et al.) describes an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The reporter molecule (e.g., fluorescein dye) is separated from the quencher molecule (e.g., rhodamine dye) by at least about 15 nucleotides, more preferably at least about 18 nucleotides. In one embodiment, the oligonucleotide probe is immobilized on a solid support either directly or by a linker.
What is clearly needed however are reagents adapted to provide further options in the course of oligonucleotide synthesis, such as longer spacers and/or the ability to be used as either the support or as an amidite. Such longer spacers can be used, for instance, to optimize the binding of biotin to avidine, or to reduce steric interference of such labels when double stranded oligonucleotides (one or both of which strands may include such labels) are hybridized to each other.
The present invention provides a new labeling reagent for use in oligonucleotide (xe2x80x9coligoxe2x80x9d) synthesis, as well as a method of preparing such a labeling reagent, a method of using such a reagent for synthesizing a labeled oligonucleotide, and an oligonucleotide prepared using such a reagent. Depending on its conditions of preparation and/or use, a reagent of the present invention can be used to label either the 3xe2x80x2 or 5xe2x80x2 termini of a synthesized oligonucleotide, and/or for one or more positions along the oligonucleotide.
The labeling reagent is preferably prepared as the condensation product of a tritylated hydroxyacid and a diamine, in a manner that provides the resulting linear reagent backbone with one or more label attachment sites and one or more sites for attaching the reagent to a support or amidite. The labeling reagent is more preferably prepared by a reaction scheme that involves the initial preparation of a DMT-hydroxyacid intermediate in the manner provided herein.
The invention therefore provides a labeling reagent useful in making labeled oligonucleotides, the reagent preferably comprising the reaction (e.g., condensation) product of a DMT-hydroxyacid and a diamine, wherein the reaction product provides at least one secondary hydroxyl group. In one particularly preferred embodiment, the hydroxacid itself provides at least one internal amide bond prior to condensation with a diamine. The resulting reagent provides the secondary hydroxyl group residue, either attached to a solid support or converted to a phosphoramidite, and further provides one (primary or secondary) amine group residue having a suitable label or protecting group attached thereto.
In one preferred embodiment, the invention provides a reagent having the following structure selected from:
R1xe2x80x94Oxe2x80x94CH2xe2x80x94CH(Oxe2x80x94R2)xe2x80x94CH2xe2x80x94NHxe2x80x94COxe2x80x94(CH2)mxe2x80x94COxe2x80x94NHxe2x80x94(CH2)nxe2x80x94NHxe2x80x94R3xe2x80x83xe2x80x83(Structure 1)
R1xe2x80x94Oxe2x80x94(CH2)nxe2x80x94NHxe2x80x94COxe2x80x94(CH2)mxe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH(Oxe2x80x94R2)xe2x80x94CH2xe2x80x94NHxe2x80x94R3xe2x80x83xe2x80x83(Structure 2a)
R1xe2x80x94Oxe2x80x94(CH2)nxe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH(Oxe2x80x94R2)xe2x80x94CH2xe2x80x94NHxe2x80x94R3xe2x80x83xe2x80x83(Structure 2b)
and
R1xe2x80x94Oxe2x80x94(CH2)3xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH(OR2)xe2x80x94CH2xe2x80x94NHxe2x80x94COxe2x80x94(CH2)mxe2x80x94COxe2x80x94NHxe2x80x94(CH2)nxe2x80x94NHxe2x80x94R3xe2x80x83xe2x80x83(Structure 3)
wherein:
R1 is 4,4xe2x80x2-dimethoxytrityl (xe2x80x9cDMTxe2x80x9d), 4-monomethoxytrityl (xe2x80x9cMMTxe2x80x9d) or any other hydroxyl protecting group stable to oligonucleotide synthesis conditions;
R2 is selected from the group consisting of: 
and salts thereof (i.e., phosphorous groups for use in providing coupling reagents), wherein R5 and R6 are independently selected from the group consisting of C3 to C10 branched alkyl, C1 to C12 unbranched alkyl, and cyclic hydrocarbons, and Y is any phosphate protecting group. In a preferred embodiment, R5xe2x95x90R6CH(CH3)2. Optionally, R2 can include a cleavable (under conventional oligonucleotide cleavage conditions) or noncleavable linkage to (and including) a suitable support such as a controlled pore glass (CPG) support, or to (and including) a glass or polymeric support for preparing an oligonucleotide array, or to (and including) an alkylamine CPG support wherein alkyl is 1 to 50 carbon atoms and isomeric forms thereof, or to (and including) a chemically modified CPG, to or (and including) a suitable polymer, such as polystyrene or divinylbenzene;
Also, in structures (1) through (3) above, R3 is a label or reporter molecule (occasionally referred to collectively herein as xe2x80x9clabelxe2x80x9d), which is stable under the conditions of oligonucleotide synthesis, preferably selected from the group consisting of biotin, fluorescein, rhodamine, 4-(4xe2x80x2-Dimethylamino-phenylazo)benzoic acid (xe2x80x9cDabcylxe2x80x9d); 4-(4xe2x80x2-Dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride) (xe2x80x9cDabsylxe2x80x9d); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid (xe2x80x9cEDANSxe2x80x9d); Psoralene derivatives, haptens, cyanines, acridines, fluorescent rhodol derivatives, cholesterol derivatives, ethylenediaminetetraaceticacid (xe2x80x9cEDTAxe2x80x9d), and derivatives and analogs thereof; as well as radioactive labels;
m=is between 2 and 4, inclusive, and can be selected by the number of methylene groups in the cyclic anhydride used in making the hydroxyacid intermediate; and
n=is between 2 and 100, inclusive, and more preferably between 2 and 20, inclusive, and can be selected by the number of methylene groups present in the precursor diamine, hydroxyacid, or aminoalcohol moieties. In case of ethyleneglycol diamines and ethyleneglycol amino alcohols, methylene groups can be replaced by corresponding ether bonds.
The secondary hydroxyl group provided by such a reagent can be used or protected in any suitable fashion. In one preferred embodiment the hydroxyl group is used to attach the reagent to a solid support in order to generate a solid support resin for use in oligonucleotide synthesis. The resulting resin, in turn, can be used to support the synthesis of an oligonucleotide that incorporates a residue of the reagent (including any label attached thereto) at the 3xe2x80x2 terminus of the newly synthesized oligonucleotide. In one suitable mechanism, for instance, the hydroxyl group can be succinylated (converted to an ester) by reaction with succinyl anhydride, in order to cleave the anhydride, forming a carboxylic acid group. The carboxylic acid, in turn, can be coupled to a group such as an amine group provide by a support such as CPG, in order to form an amide linkage between the reagent and support. The resulting support can be used as a 3xe2x80x2 labeling reagent in solid phase oligonucleotide synthesis.
Alternatively, the hydroxyl group can be converted to a phosphorylating group, in order to permit the resultant reagent to be used in solution and in the course of oligonucleotide synthesis for 5xe2x80x2 and internal labeling. Preferred phosphorylating groups include phosphoramidite groups, and particularly, cyanoethylphosphoramidite groups.
A labeled phosphoramidite of this invention, and particularly those having long spacers, can be used for attaching a label at the 3xe2x80x2-end of an oligonucleotide in a manner that is sufficiently stable towards regular cleavage conditions typically used in the deprotection and/or cleavage of oligonucleotides. Such stable linkages can be obtained, for instance, when an aminated support is converted to a hydroxylated support, which upon reaction with the amidite reagent of this invention forms the phosphite bond. The phosphite bond, in turn, is typically oxidized to a phosphotriester and upon deprotection steps is converted to a stable phosphodiester. The provision of a hydroxylated support, or conversion of an aminated support to a hydroxylated support, can be accomplished by any suitable means, e.g., by treating an aminated support with gamma-butyrolactone under conditions (e.g., heating) suitable to convert the amine functionalities to corresponding 3-hydroxypropyamido functionalities. Such a process can be performed using aminated supports such as aminopropyl-CPG or long chain alkylamine-CPG (LCAA-CPG) supports that are commercially available, as well as aminated polystyrene supports.
Given the present description, those skilled in the appropriate art will be able to employ a variety of suitable synthetic strategies for such purposes, e.g., as described in Vu, et al., Nucleosides and Nucleotides 12(8):853-864 (1993), the disclosure of which is incorporated herein by reference. A DMT-hydroxyacid intermediate can be prepared in a reaction between an aminoalcohol and a cyclic anhydride, typically succinic anhydride. To the extent any such hydroxyacids are not commercially available, they can be prepared in the manner described herein.
An amine group, for use as the R3 substituent in a reagent of the present invention, can itself be functionalized with a variety of labels, e.g., selected from the group consisting of biotin, fluorescein, rhodamine, 4-(4xe2x80x2-dimethylamino-phenylazo)benzoic acid (xe2x80x9cDabcylxe2x80x9d); 4-(4xe2x80x2-dimethylamino-phenylaz acid (sulfonyl chloride) (xe2x80x9cDabsylxe2x80x9d); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid (xe2x80x9cEDANSxe2x80x9d); Psoralene derivatives, haptens, cyanines, acridines, fluorescent rhodol derivatives, cholesterol derivatives; ethylenediaminetetraaceticacid (xe2x80x9cEDTAxe2x80x9d) and derivatives thereof; and radioactive labels. The labeling reagents of this invention can include succinylated labeling reagents (and other ester derivatives), e.g., in a form coupled to a support for 3xe2x80x2-end labeling, or in the form of a phosphoramidite labeling reagent for 5xe2x80x2-end labeling or for internal labeling of an oligonucleotide.
The labeling reagents described herein can be prepared using materials that are commercially available or that can be readily synthesized, given the present description. In turn, the extent of spacing providing by a reagent can be varied as desired, e.g., by the selection and use of a wide variety of diamines that are commercially available and inexpensive (e.g., as used in the production of Nylon).